In cardiac Purkinje fibers two cellular processes have been observed to increase cytoplasmic (CA++) during excitation-contraction coupling. The following hypotheses have been formulated: The first process is CA++ entry through surface membrane CA++ channels; this results in an initial rapid rise in the indicator response (aequorin luminescence). The second process is release of CA++ from stores; accumulation of released CA++ results in a second peak or a plateau in the aequorin signal. The initial goal of the proposed research will be to more rigorously test these hypotheses under new experimental conditions. Membrane potential will be controlled (voltage clamp), in order to observe the influence of voltage on these processes, and in order to gain a degree of control over the intracellular (CA++) transient not attainable with action potentials. The aequorin signal under these conditions will be compared with known observed, and putative features of surface membrane CA++ channels (time and voltage dependence), CA++ stores (depletion, loading, delayed reavailability), and Na/Ca exchange. The second major goal of the proposed research is to evaluate the hypothesis that CA++ induced release of CA++ is a physiological mechanism of excitation-contraction coupling in intact cells. This will be done primarily through manipulation of the (CA++) transient by control of membrane voltage. A critical question will be: In a particular response, does the magnitude of cyroplasmic CA++ accumulation due to the second process depend on the magnitude of the first (when voltage is controlled and under steady state conditions)? The physiological importance of answering this question will be enhanced if the underlying cellular processes can be identified as above. Finally, the mechanism of the positive inotropic and toxic dysrhythmic effects of a cardiotonic steriod, acetylstrophanthidin, will be examined. The proposed research will be part of the long term objective to directly observe intracellular CA++ and understand its role in cardiac physiology; in regulation of contractility by membrane potential effects, hormones, neurotransmitters and drugs, and in electrophysiology per se, as in dysrhythmias associated with CA++ overloaded states.